Evaluation of methods for improved detection of Cryptosporidium spp. in mussels ( Mytilus californianus)

• Published in: Journal of microbiological methods Vol. 65; no. 3; pp. 367 - 379
• Main Authors: Miller, Woutrina A., Gardner, Ian A., Atwill, Edward R., Leutenegger, Christian M., Miller, Melissa A., Hedrick, Ronald P., Melli, Ann C., Barnes, Nicole M., Conrad, Patricia A.
• Format: Journal Article
• Language: English
• Published: Shannon Elsevier B.V 01.06.2006; Elsevier Science
• Subjects: Animals; Bacteriological methods and techniques used in bacteriology; Bacteriology; Biological and medical sciences; Bivalve; Bivalvia; Cryptosporidium; Cryptosporidium - classification; Cryptosporidium - genetics; Cryptosporidium - growth & development; Cryptosporidium - isolation & purification; DNA, Protozoan - analysis; Fluorescent Antibody Technique, Direct; Fundamental and applied biological sciences. Psychology; Immunomagnetic Separation; IMS; Microbiology; Mussel; Mytilus - parasitology; Mytilus californianus; Oocysts - genetics; Oocysts - isolation & purification; Polymerase Chain Reaction; Real-time PCR; Sensitivity and Specificity; Systematics; Taq Polymerase; Waterborne pathogen; Cryptosporidium; Mussel; Real-time PCR; Bivalve; Waterborne pathogen; IMS; Protozoa; Evaluation; Apicomplexa; Mytilus californianus; Method; Real time; Polymerase chain reaction; Bivalvia; Magnetism; Invertebrata; Mollusca; Detection; Immunofluorescence; Waterbome pathogen
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/j.mimet.2005.08.011

Bivalve molluscs concentrate Cryptosporidium oocysts from fecal-contaminated aquatic environments and are therefore useful in monitoring water quality. A real-time TaqMan polymerase chain reaction (PCR) system was developed to allow for large scale quantitative detection of Cryptosporidium spp. in mussels ( Mytilus californianus). The TaqMan sensitivity and specificity were compared to conventional PCR and direct immunofluorescent antibody (DFA) assays, with and without immunomagnetic separation (IMS), to identify the best method for parasite detection in mussel hemolymph, gill washings and digestive glands. TaqMan PCR and two conventional PCR systems all detected 1 or more oocysts spiked into 1 ml hemolymph samples. The minimum oocyst detection limit in spiked 5 ml gill wash and 1 g digestive gland samples tested by TaqMan PCR and DFA was 100 oocysts, with a 1 log 10 improvement when samples were first processed by IMS. For tank exposed mussels, TaqMan and conventional PCR methods detected C. parvum in < 5% of hemolymph samples. No gill washings from these same mussels tested positive by TaqMan PCR or DFA analysis even with IMS concentration. All methods detected the highest prevalence of C. parvum-positive samples in digestive gland tissues of exposed mussels. In conclusion, the most sensitive method for the detection of C. parvum in oocyst-exposed mussels was IMS concentration with DFA detection: 80% of individual and 100% of pooled digestive gland samples tested positive. TaqMan PCR was comparable to conventional PCR for detection of C. parvum oocysts in mussels and additionally allowed for automated testing, high throughput, and semi-quantitative results.